Brazil, since the beginning of the '90s, stays as the major world producer of orange, being responsible for 80% of the international trade of concentrated and frozen orange juice. In order to obtain this production, estimated in US$900 million, about US$410 million of input is spent, leading to approximately US$1.5 billion in transactions involving the marketing of citrus products, such as juices and fresh fruit (FNP Consultoria & Comércio, 2008. Available at <http://www.fnp.com.br/agricultura/citros/prod_area_laranja.php> Access in Sep. 23, 2008). The second major producer is the United States, followed by Mexico, China and Spain. Spain and the United States are the main exporters of fresh fruit, which corresponds, respectively, to 38 and 18% of the world exports (BOTEON, M.; VIDAL, A. J. Citricultura no Brasil e na Flórida. Citricultura Atual, n. 23, p. 3, 2001).
The state of São Paulo (Brazil) has around 34.2 million of plants under development and 163.5 million citric plants in production, reflecting more than 80% of the national production of citric fruits. Currently, it is responsible for 97% of the Brazilian exports, being the grand dynamic core of the Brazilian citrus complex. This market is responsible for approximately US$1.5 billion of currency for the country, state and cities and employs 400 thousand people. The citrus park of São Paulo together with a region in Minas Gerais known as “Triângulo Mineiro” presents 198 million trees. The harvest of oranges is performed throughout the year due to the composition of the varieties such as “Hamlin”, “Lima”, “Pêra”, “Seleta”, “Bahia”, “Natal”, “Valência” and “Folha Murcha”. Among these, some are appropriate for the production of juice, such as “Pêra”, which represents 38% of the total of the trees, followed by “Valência”, “Natal” and “Hamlin”, with respectively 17, 25 and 7% (BARROS, M. H. C.; BOTEON, M. O Brasil é o único país paie produz o ano inteiro. Hortifruti Brasil, v. 3, p. 21, 2002.).
With the advance of the technology in the field, the plants started to be multiplied by grafting, which rendered great advantages regarding the precocity and uniformity of the orchards. However, at the same time it reduced the variability, making the culture a constant target to several pests and diseases which, when facing favorable conditions to their development, are capable of causing irreversible damage. The phytosanitary costs, along with the fertilizers, are higher than 60% of the cost of production. The quality and the quantity of the citric fruits are frequently threatened due to the damage left in the plants, which, depending on the intensity of the attack, can make it unproductive or lead to its eradication.
In the middle of 2004 a new disease was reported in the orchards from São Paulo. A devastating disease without cure originated from Asia and Africa. Huanglongbing (HLB), also known as Greening, is currently the most feared disease among the orange producers due to the fact that it does not possess cure or treatment, since it has a high rate of dissemination and affects all the commercial varieties of oranges. The most efficient solution until this moment in order to minimize the losses is the eradication of the plants as soon as a positive diagnosis for the disease is confirmed.
The Greening disease has as causing agent a bacterium that lives in the phloem of the host plant, being known as Candidatus Liberibacter. There are three types of bacteria related to Greening, Candidatus Liberibacter africanus, Candidatus Liberibacter asiaticus and Candidatus Liberibacter americanus, being the latter the main causing agent in the state of Sâo Paulo. The symptoms of Greening are visually the same, disregarding where the disease occurs. Initially, the symptoms appear in the branches, which are evident due to the yellow coloration contrasting with the green coloration of the leaves from the non affected branches. The leaves present pale yellow coloration, with green sectors, rendering irregular and asymmetric spots (mottled). The fruit becomes deformed and asymmetric. The white part of the peel, in some cases, presents thickness higher than normal. The reduction in the size of the fruit and intense fall also occur.
Due to the lack of a cure, the Greening disease is, currently, the most feared diseases in the culture of citrus. It was already responsible for the eradication of more than 2 million citrus trees and the great majority of them were in the season of higher productivity.
In particular, the culture of citrus of the state of São Paulo presents some peculiar characteristics that lead to a high vulnerability of the culture to the occurrence of epidemics of known diseases such as Decline disease, and of new diseases, such as the Citrus Sudden Death (CSD) and Greening disease, rendering important consequences to the sustainability of the agribusiness:                1) The orchards of citrus of the state of São Paulo occupy a nearly continuous area of approximately 615,300 ha (FNP. Citrus. AGRIANUAL 2003: anuário da agricultura brasileira. FNP consultoria & agroinformativos, São Paulo, p. 295-314, 2003.), without great variations of relief or physical barriers and with high traffic of people between them;        2) The citric plants are perennial and, therefore, are exposed throughout the year and for several years to the attack of pests and diseases. Besides that, the emission of new shoots can be observed during a great part of the year, which confers a continuity of susceptible tissue and of inocule between the cultivation seasons;        3) The orchards of the state of São Paulo present a very low genetic variability. Only four varieties of sweet orange (Citrus sinensis Osbeck): ‘Pêra-Rio’, ‘Natal’, ‘Valencia’ and ‘Hamlin’, propagated vegetatively through grafting, represent 92% of the Brazilian citrus culture. The rootstock used in approximately 85% of the trees is the ‘Cravo’ lime (C. limonia Osbeck) due to its rusticity, vigor conferred to the canopy and resistance to the hydric deficiency of the north and northwest of the state.        
Currently, there are some diagnosis methods for Greening disease. The most common diagnosis is performed through the visual inspection of leaves presenting the symptoms of the disease. The control of the Greening disease in the state of São Paulo and in the “Triângulo Mineiro” has been achieved through periodic visual inspections performed by technicians of the Fundecitrus or people trained by them. This method has as its characteristics a high rate of subjectivity and a high percentage of error, besides the fact that the disease is only diagnosed after the expression of the symptoms, which is, after a long period of asymptomatic phase of the tree (approximately 8 months). During the asymptomatic phase of the disease, the infected tree is an invisible source of dissemination of the disease leading to a very large delay in the eradication of the tree. However, after the visualization of the symptoms, the uprooting is the only current matter of control of the disease.
The document JP2006267092-A described a detection kit for Greening disease in early stages, based on the recognition of the drop in the manganese and iron concentrations in the tissues of the infected tree. The detection procedure includes the obtaining of a water soluble extract of plant material containing a buffer system, followed by analysis of absorption spectrophotometry. The disease determination, however, is unspecific, since the low manganese and iron concentrations in a plant can be due to several physiological causes, including deficiency of these minerals in the plant nutrition. In an analogous manner, the document JP2004264101-A claims the detection of the Greening disease in plant extracts via the analysis of the starch concentration through the iodine reaction, thus presenting, the same disadvantage in the physiological unspecificity of the deficiency.
A method described in the document WO2007021485-A2 promises the detection of Greening causing bacteria, among the detection of several other pathogens and chemical contaminants from plants and animals, using the Raman Effect of applied polarized radiation scattering. However, the detection is conditioned to aqueous samples that are submitted to a complex treatment that includes drying over colloidal metallic surface, followed by contact with ultra filtering membrane.
The Apta Citrus, affiliated to the Instituto Agronômico de Campinas (IAC), is the only center of the state of São Paulo to provide technical reports for the diagnosis of Greening disease. This report is based in a technique called PCR (Polymerase Chain Reaction), which consists in the genetic analysis of the leaves in search for the DNA of the Greening causing bacteria. However, the detection of the bacteria DNA is only possible in symptomatic leaves (mottled), and even then, it can fail in 10% of the cases. For the diagnosis in asymptomatic leaves (without visible signs of the disease), the IAC uses a more complex variation of the technique called real time PCR, which its sensitivity is at least 1000 times higher than the conventional technique. However, in function of its complexity, the real time PCR has not been used in routine exams, only for scientific work. Even though the PCR technique is highly efficient, it is costly and laborious (BOVÉ, J. M. History, etiology, field identification, transmission, and world distribution of huanglongbing: a destructive, newly-emerging, century-old disease of citrus. Huanglongbing Greening International Workshop, v. 1, p. 1, 2006) The cost of the analysis per sample, using the conventional PCR, is around US$25.00 and a report can take around 20 days. The use of PCR in large scale is economically unviable; besides the fact that it produces an infestation mapping that is very temporally delayed. On the other hand, the document CN1824802-A claims the detection of the asymptomatic trees in only 3 h after the sampling, but it is restricted to the detection caused by the bacteria Candidatus Liberobacter asiaticum Jagoueix and to the citrus of the mandarin type.
It is in this moment that the fluorescence becomes a relatively simple and efficient dispositive that can make a difference. The fluorescence is a technique that allows the detection of chemical alterations that occurs in the leaves of plant affected by Greening and by other diseases prior the perception of their visual manifestations.
In the document BR200201249-A, the auto-fluorescence of the leaves, fruits and branches allows the detection of the citric canker in asymptomatic citrus, after the exposition to light sources with short wavelength. However, the technique is limited to the detection of this disease, caused by the bacteria Xanthomonas axonopodis pv. citri. 
This invention developed in the Embrapa Instrumentação Agropecuária has as its objective to protect a new diagnosis methodology and apparatus for diagnosing stresses and diseases in higher plants, such as the citrus Greening disease. A work previously performed in the Laboratório de Óptica e Lasers da Embrapa Instrumentação Agropecuária LOLEIA (document PI 05059757-7), demonstrated the possibility of distinction of healthy leaves from sick ones, and also, of distinction of diseases (CSD and Decline) through the ratio of the maximum of the fluorescence emission spectrum using commercial spectrometers. The basic idea of the proposed methodology was the following: when a plant reaches imbalance, being caused by stress or by disease, changes in its metabolism occur that lead to an alteration of the chemical composition of its organs. This chemical change leads to a change in the physical properties, such as, for example, reflectance and fluorescence of the leaves. Based in this hypothesis, it was developed a laboratory study using commercial apparatus, regarding the fluorescence emission of the leaves of healthy and sick citrus leaves. The possibility of distinction of healthy trees, tress with Decline or CSD was observed through the ratio of fluorescence emission peaks, method described in PI 05059757-7.
In the patent application PI 05059757-7, the idea of a portable module that performs spectroscopic measurements in leaves was protected for the diagnosis of citrus sudden death with excitation using bulb, led or laser in the range of 420 to 480 nm. In 2006, the LOLEIA developed a system using laser induced fluorescence spectroscopy (LIFS), similar to the one proposed in the document PI 05059757-7, with a probe constructed with optical fibers and detection system constituted of a set of calibrated photodiodes that permits the obtaining of the emission spectrum of the leaf in an extremely quick manner (˜1 s) and excitation with laser at 470 nm. With this new system, it became possible to analyze a large volume of samples in reduced time. At that time, Greening disease was already considered the most severe disease of the Brazilian citrus sector, and, therefore, the LOLEIA initiated research in order to evaluate the possibility of diagnosis of Greening using fluorescence spectroscopy. With the preliminary results, it could be observed that the best distinction for the Greening disease was observed with other laser with 561 nm wavelength excitation. With the reformulated portable module, a discerning study to evaluate the possibility of the diagnosis of Greening through the leaves was performed. From this study, a new disease diagnosis methodology different from the one of the patent application PI 05059757-7 (for CSD) was resulted. Even though the current methodology uses similar apparatus protected for the CSD, with different excitation wavelength, the system as a whole is totally different and main differences will be discussed below.
In the current system, the ratios of peaks are not used as in the document PI 05059757-7. It was developed a software to collect the fluorescence spectra and to perform an statistical treatment of them (FIGS. 1 and 2) in order to evaluate in the most discerning manner all the emission spectrum to obtain a higher number of information that enables a more precise diagnosis of the diseases.
After being treated by the Principal Component Analysis (PCA) statistical method, agglomerations in tridimensional graphics of the first main components were clearly defined as shown in FIG. 3.
As the system became very sensitive, it also distinguished the variety of the canopy and of the rootstock, reason why another patent application was elaborated with this theme, the “Método e Equipamento para Certificação de Mudas de Citros” (Method and Equipment for Certification of Citrus Seedlings). This way, for each combination of canopy and rootstock, a system calibration process was performed. For each combination of canopy and rootstock, a set of samples containing healthy and sick leaves were used in order to provide information to the system of typical spectra of each class of samples. In general, the following classes were evaluated: healthy samples, sick samples presenting Greening in the symptomatic phase, sick samples presenting Greening in the asymptomatic phase, with CSD and with Decline.
Using tools of machine learning, another software was developed in such way that the calibration database was used in a manner that the system could be capable of measuring a totally unknown leaf and classifying the tree status as healthy or sick, and, if sick, identifying the disease. In the case of Greening disease, it was possible to identify the disease in the symptomatic phase as well as in the asymptomatic phase. This result shows the great potential of the technique for enabling the performance of early diagnosis of the disease.
In the patent of CSD, the spectroscopy analysis was only performed after the obtaining of a map of the orchard through fluorescence images, which performed a distinction of healthy trees from sick ones. In the current patent, the diagnosis is performed using only the fluorescence spectroscopy.
Therefore, different from the patent PI 05059757-7, the apparatus as it was protected per se is not capable of performing an automatic diagnosis. The complete system needs the acquisition software and statistical treatment of the data, calibration database and classifying software. This way, in this patent, the protection of the complete system composed by the apparatus for performance of fluorescence spectroscopy measurements, the acquisition software and statistical treatment of the data, database and classifying software for the performance of the diagnosis of Greening through the citrus leaves is requested.